C/EBPa is a transcription factor that is critical for differentiation to granulocytes, hepatocytes, adipocytes and lung cells. It appears to function as a tumor suppressor and its dysregulation has been shown to play a role in Acute Myelogenous Leukemia (AMI). The strict regulation of transcription factor expression may play a critical component in the development of leukemia and other diseases. Thus, we initiated studies to isolate and characterize human CEBPa regulatory elements in myeloid cells. We used DNase I hypersensitivity assays to identify a 131 bp region at +33 kb downstream of the CEBPa gene that is highly conserved across mammalian species and transcriptionally active during granulocytic differentiation. We have shown by luciferase assays that this region functions to enhance luciferase activity driven by the C/EBPa promoter by up to 12-fold over expression vectors containing the promoter alone. Lastly, we have shown by gel shift analysis that this region can bind the transcription factors PU.1 and YY1. Thus, this proposal seeks to test the hypothesis that this and other conserved elements downstream of the CEBPA gene act to regulate C/EBPa expression and that these elements are required for C/EBPa expression during granulopoietic differentiation. We will test this hypothesis in 2 Aims. First we will determine whether the 131 bp putative regulatory element is-sufficient to act as an enhancer of C/EBPa expression during differentiation of hematopoietic cells. Second, we will determine whether YY1, PU.1, or other proteins regulate C/EBPa through the ORE during granulopoiesis.